The autophagy pathway directs the principle recycling center of the cell, where long-lived proteins, bulk cytoplasmic material and damaged organelles (e.g., mitochondria) are engulfed by double-membraned vesicles coined autophagosomes, which then fuse with the lysosome that degrades this cargo to recoup building blocks and energy under times of stress or nutrient deprivation. Accordingly, autophagy is necessary for cellular homeostasis, and defects in the pathway leads to various pathologies, including neurodegenerative diseases and myopathies. Importantly, we have shown that impairing autophagy augments the efficacy of anti-cancer drugs and can overcome resistance. Though there is much interest in generating antagonists and agonists of specific components of the autophagy pathway, currently such agents are lacking. The autophagy pathway is controlled by a conserved serine/threonine kinase coined Ulk1 (UNC-51-like kinase-1). Our Multi-PI research team has shown that Ulk1 kinase activity is essential for control of the autophagy pathway that Atg13 is a bona fide substrate of Ulk1 and that Ulk1-directed phosphorylation of Atg13 on S318 is essential for autophagy. Importantly, we have developed reagents that allow us to monitor Atg13 phosphorylation by ULK1, intracellular Ulk1 kinase activity, rates of autophagic flux and the maturation and fusion of autophagosomes with lysosomes. Given the importance of Ulk1 as a target, we will enable, develop and optimize a focused set of biochemical and cell-based assays to identify and characterize small molecule inhibitors of Ulk1. In Specific Aim 1 we will develop a novel homogenous high-throughput screening (HTS) compatible assay based on the phosphorylation of full-length human Atg13 by full-length Ulk1. This assay uses a bead-based proximity format ideally suited for miniaturization and high-throughput screening (HTS). Once validated, this assay will be submitted to the Molecular Libraries Production Centers Network (MLPCN) to identify probes of Ulk1 by performing a HTS-campaign against the Molecular Libraries Small Molecule Repository (MLSMR) compound collection. Identified and confirmed 'hits' will be counter-screened against a panel of select kinases to triage compounds to drive medicinal chemistry efforts. To assess lead declared molecules, in Specific Aim 2 we will develop a cell-based assay that measures Atg13 phosphorylation and that will provide a quantitative measure of intracellular potency. This assay will drive structure activity relationshi (SAR) of Ulk1 cell-based inhibitor activity in subsequent rounds of medicinal chemistry supported by the MLCPN. In Specific Aim 3 we will develop a novel cell-based assay that allows one to determine rates of autophagic flux in real time in living cells. This assay will allow us to quantify effects of our top Ulk1 inhibitors in response to different cues that activate the autophagy pathway. Collectively, these studies will provide a comprehensive set of decision making tools that will greatly facilitate the development of potent and selective, cell-penetrant Ulk1 inhibitors that can be used to interrogate the biological role(s) that Ulk1 plays in normal an pathological states.